Evidence That a Phosphatidylinositol 3,4,5-Trisphosphate-binding Protein Can Function in Nucleus

Abstract

PIP 3 BP is a phosphatidylinositol 3,4,5-trisphosphatebinding protein (PIP 3 BP) abundant in brain, containing a zinc finger motif and two pleckstrin homology (PH) domains. Staining of rat brain cells with anti-PIP 3 BP antibody and determination of localization of PIP 3 BP fused to the green fluorescent protein (GFP-PIP 3 BP) revealed that PIP 3 BP was targeted to the nucleus. Targeting was dependent on a putative nuclear localization signal in PIP 3 BP. Generation of PIP 3 in the nucleus was detected in H 2 O 2 -treated 293T cells, nerve growth factor (NGF)-treated PC12 cells, and platelet-derived growth factor (PDGF)-treated NIH 3T3 cells. Translocation of phosphatidylinositol 3-kinase (PI 3-kinase) to the nucleus and enhanced activity of PI 3-kinase in the nucleus fraction were observed after H 2 O 2 treatment of 293T cells, suggesting that PI 3-kinase can be activated in the nucleus as well as in the membrane after appropriate stimulation of the cells. Co-expression of the constitutively active PI 3-kinase with PIP 3 BP resulted in exportation of the protein from the nucleus to the cytoplasm, suggesting that PIP 3 BP can function as a PIP 3 -binding protein in the intact cells. These results imply that there may be an unknown function of PI 3-kinase in the nucleus.Phosphatidylinositol 3-kinase 1 is an enzyme that is activated immediately after growth factor or differentiation factor stimulation of the cells (1) and that generates second messengers, phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) and phosphatidylinositol 3,4-bisphosphate (PI 3,4-P 2 ) (2-5). These 3Ј-phosphorylated phosphoinositides can activate serine, threonine kinases such as PKB/Akt, PKCs, and PDKs (6 -9). They are also suggested to be involved in other events such as rearrangement of cytoskeleton and vesicle transport because these phenomena are sensitive to the PI 3-kinase inhibitors and dominant negative mutants of PI 3-kinase (10). Recently, it was reported that the 3Ј-phosphorylated phosphoinositides can activate guanine nucleotide exchanging factors of Rac and Arf, small G proteins involved in actin rearrangement and vesicle transport, respectively (11,12). Therefore, G proteins as well as kinases are downstream of PI 3-kinase.We have identified PIP 3 BP as a PIP 3 -binding protein, using a PIP 3 analogue column (13). It is abundant in brain, implying that it may be involved in the function of nerve systems. PIP 3 BP binds to PIP 3 but not to PI 3,4-P 2 or phosphatidylinositol 4,5-bisphosphate (PI 4,5-P 2 ). It has a zinc finger motif homologous to that of Arf-GTPase activating protein (GAP) and two PH domains. Both PH domains are shown to be involved in binding to PIP 3 . Another PIP 3 -binding protein, centaurin ␣, is highly homologous to PIP 3 BP (14). No GAP activity to Arf has been detected in either protein. Although the binding of centaurin ␣ and PIP 3 BP to PIP 3 was specific, the role of the protein is unclear. To address this question, we determined the intracellular localization by immunological techniques, using mono...

The role of PTEN signaling perturbations in cancer and in targeted therapy

“…The second messenger PIP3 binds to a subset of pleckstrin homology domain-containing proteins, enabling the plasma membrane recruitment and activation of target proteins (Frech et al, 1997;Klarlund et al, 1997). Intriguingly, PIP3 is also found in the nucleus, along with PTEN and other pathway components, revealing a more complex view of the PTEN network (Tanaka et al, 1999;Liu et al, 2005a, b;Deleris et al, 2006;Gil et al, 2006;Li et al, 2006;Lindsay et al, 2006;Trotman et al, 2006Trotman et al, , 2007Baker, 2007;Shen et al, 2007;Chang et al, 2008). PI3K signaling induces a diverse array of cancerpromoting events including the regulation of the protein kinases PDK1 and AKT, which directly bind to and are activated by PIP3 (Alessi et al, 1997;Currie et al, 1999).…”

The Major Target of the Endogenously Generated Reactive Oxygen Species in Response to Insulin Stimulation Is Phosphatase and Tensin Homolog and Not Phosphoinositide-3 Kinase (PI-3 Kinase) in the PI-3 Kinase/Akt Pathway

“…It has been demonstrated that the activity of PI-3 kinase, the level of 3-phosphorylated PIs, and the activity of Akt are increased in cells exposed to H 2 O 2 (Konishi et al, 1997;Shaw et al, 1998;Sonoda et al, 1999;Tanaka et al, 1999;Qin et al, 2000). This H 2 O 2 -mediated activation of PI-3 kinase/Akt pathway is considered to be the result of reversible oxidation and inactivation of protein tyrosine phosphatase (PTP) family protein such as PTEN that have a critical cysteine residue in the active site (Denu and Tanner, 1998;Lee et al, 1998;Meng et al, 2002).…”

“…Because PTEN has a critical role in antagonizing PI-3 kinase signaling pathways, it might be expected that PTEN would be the target of complex control mechanisms. Only limited studies have addressed this issue, but evidence is now emerging for functional regulation of PTEN at the level of protein stability and localization, as well as transcription of the PTEN gene (Adey et al, 2000;Vazquez et al, 2000Vazquez et al, , 2001Kurose et al, 2001;Bastola et al, 2002).It has been demonstrated that the activity of PI-3 kinase, the level of 3-phosphorylated PIs, and the activity of Akt are increased in cells exposed to H 2 O 2 (Konishi et al, 1997;Shaw et al, 1998;Sonoda et al, 1999;Tanaka et al, 1999;Qin et al, 2000). This H 2 O 2 -mediated activation of PI-3 kinase/Akt pathway is considered to be the result of reversible oxidation and inactivation of protein tyrosine phosphatase (PTP) family protein such as PTEN that have a critical cysteine residue in the active site (Denu and Tanner, 1998;Lee et al, 1998;Meng et al, 2002).…”

Phosphatidylinositol 3-kinase mediates activation of ATM by high NaCl and by ionizing radiation: Role in osmoprotective transcriptional regulation

“…Some PI3K occurs in the nucleus (28,29). Stimulation of cells by nerve growth factor activates nuclear PI3K, resulting in accumulation of 3-phosphorylated phosphoinositide lipids in the nucleus (29,30). That is mediated by PLC␥, which activates PI3K Enhancer, a nuclear GTPase that increases PI3K activity (31,32).…”